Duchenne muscular dystrophy (DMD), with a prevalence of 1 boy out of 3500, is the most widely spread genetic disease (Voisin, 2004). This disease is characterized by the progressive weakening of muscles, resulting in muscular necrosis and fibrosis (Emery, 2003). An essential protein for muscle fibers stability, dystrophin, is missing or defective in muscle fibers of DMD patients. Dystrophin is a 427-kDa protein which connects the actin cytoskeleton to the cellular membrane (Bogdanovich et al. 2004). The function of this protein is to maintain the stability of the cellular membrane in order to support the stresses induced during muscular contractions (Carpenter et al. 1990). The absence of dystrophin increases the vulnerability of muscle fibers during their contraction. Consequently, constant muscular repair will be necessary, which will result in the premature senescence of myoblasts.
Cellular therapy, a therapy under development to counter DMD, has shown significant therapeutic effect in several studies in mice (Chen, Li et al. 1992) and humans (Gussoni et al. 1992; Huard et al. 1992; Huard et al. 1994; Skuk et al. 2006; Skuk et al. 2007). This curative approach is regarded as a promising treatment (Skuk and Tremblay, 2000). Presently, cellular therapy consists in injecting human myoblasts in the muscles of the DMD patients. The healthy myoblasts fuse to muscle fibers of the patients and partially restore the expression of dystrophin. This expression increases the strength of the treated muscle and restores at least partially their functionality, hence improving significantly the patient's quality of life. Nevertheless, some difficulties remain to be surmounted, of which the immunizing response against the injected myoblasts, the absence of fusion of myoblasts with undamaged fibers as well as the poor migration of the myoblasts in the muscle tissue. Moreover, the culture medium presently used for myoblast expansion contains blood serum and the production processes presently used are not adequate for the production of the large number of myoblasts that would be required to treat the whole muscle mass of a patient. Constant and rapid progresses are made on these fronts, through better understanding of myoblast and myogenic cell biology, transplantation and migration, the development of protocols to alleviate immuno-rejection, and the identification of alternative cell sources, e.g. pluripotent stem cells (Skuk et al. 2002). Cellular therapy therefore seems to be on the verge of being the very first accepted therapy against DMD.
In addition to the treatment of DMD, myoblast injections are also used for the treatment of myocardial infarction and for urethral sphincter insufficiency. Indeed, the grafts of myoblasts significantly improve the contractility as well as the viability of cardiac tissues and allows 41% restoration of the normal sphincter contractility (Yiou et al. 2004; Kahn, 2006).
The nutritional needs of in vitro cultured cells comprise a vast range of molecules, of which several are found in a basal medium, which contains the essential components necessary to the cellular metabolism and osmotic balance maintenance: salts, amino-acids, vitamins, sugars, lipids, trace elements, antioxidants and pH buffer (Ham and McKeehan 1979; Butler, 1991; Mather and Barnes 1998; Davis, 2002; Naomoto et al. 2005). The basal medium must be selected considering its compatibility with the type of cells to be cultured, as well as its performance with regards to predetermined responses of interest. The role of the basal medium can therefore either be to support proliferation, differentiation or quiescence of a given cell population (Zimmerman et al. 2000). Basal media known to support myoblast expansion are DMEM, F12, RPMI1640 (Goto et al. 1999) and MCDB120 (Ham et al. 1988).
The medium generally used to culture myoblasts in vitro (standard medium, STD) is MCDB120 supplemented with 15% fetal bovine serum (FBS), 10 ng/ml basic fibroblast growth factor (bFGF), 0.39 μg/ml dexamethasone and 0.5 mg/ml bovine serum albumin (BSA). Serum is an additive which allows the non-specific proliferation of a vast range of cell types. It is prepared from plasma, the liquid fraction of blood, from which clotting factors were removed. The principal sources of sera for cell culture are bovine fetal blood (FBS), calf (CS), horse (HS) or human. FBS is the most widely used. Its functions are multiple: adhesion of the cells to a solid surface (via adhesion molecules such as fetuin, fibronectin, vitronectin), growth stimulation (growth factors, GF; cytokines; hormones), protection (antioxidants, antitoxins, proteins), buffer and nutrition. However, serum composition is not completely resolved since it is a very complex fluid, containing at least 3 000 different proteins (Omenn, 2005). Another point to consider is the quickly rising cost of serum, due to an increasing demand (500-600$/L) (Davis, 2002). Moreover, serum requires extensive quality control, it contains proliferation inhibitors, its composition varies from batch to batch, and it hinders the purification of cell culture products. Serum can also be contaminated by viruses, bacteria and prions (Jayme et al. 1988; Freshney, 2000). It is therefore advantageous to replace serum by a mixture of defined components that would not present similar problems. Serum-free media allow a better control of cell proliferation and differentiation and reduce the risk of contamination (Zimmerman et al. 2000). However, serum-free media are much more specific than serum-containing media, often only supporting the growth or differentiation of the cell type for which it has been developed. The corollary of this is therefore that most cell lineage will require the development of their own serum-free medium.
Although serum-free medium formulations for myoblast culture have previously been reported (Ham et al. 1988) or are commercially available (Skeletal muscle cell medium BULLETKIT™, CC-3160 from Lonza), the extent and rate of myoblast proliferation in these media remains much lower than in serum containing media. To this date, Applicant is not aware of any effective serum-free medium for myoblast expansion.
Consequently, the development of a safer, serum-free culture medium that would efficiently support the expansion of myoblasts and their precursors would significant and timely contribute to the advent of cellular therapy based on the use of these cells.